Abstract. Polysubstance Substance Use (PSU) is common among individuals meeting diagnosis for any substance use disorder (SUD) and is defined as the use of two or more addictive drugs. These drug combinations are associated with increased short- and long-term mental and physical health concerns. Due to the high degree of variability in PSU patterns, limited data are available regarding the molecular mechanisms underlying PSU vulnerability. The overall goal of this project is to use novel approaches based on genome-wide data to dissect the fundamental biology of polysubstance abuse and addiction. In the R21 phase of this proposal, we will conduct heritability and high-resolution cross-phenotype polygenic risk score (PRS) analyses of probabilities of PSU classes in three moderately-large study populations characterized by a high degree of PSU, our Yale-Penn cohort, the SAGE (Study of Addiction: Genetics and Environment) cohort, and the ICGHD (International Consortium on the Genetics of Heroin Dependence) cohort. The PSU pattern will be identified applying latent class analysis (LCA) and a multinomial logistic regression procedure to substance use data available from the SSADDA (Semi-structured Assessment for Drug Dependence and Alcoholism; Yale-Penn Cohort), the SSAGA (Semi-Structured Assessment for the Genetics of Alcoholism; SAGE cohort), and the SSAGA-OZ (SSAGA ? Australia; ICGHD cohort). We expect that the R21-Phase analyses will identify heritable PSU patterns and gene sets associated with them, providing the background necessary to investigate PSU in other molecular paradigms. In the R33 phase of the project, we will test the R21-phase results with respect to two different settings: 1) longitudinal PSU data; and 2) PSU-induced epigenetic changes. Re-contacting a sub-sample of the Yale-Penn cohort, we will be able to assess the trajectory of PSU patterns and the consequences of PSU and to test whether the genetic factors associated with the initial PSU status predict the PSU trajectories and consequences. Similarly, we will also test whether heritable PSU correlates with epigenetic changes and whether these mediate health outcomes. The expected results of the R33 phase will provide multiple findings related to the biology of PSU that can improve clinical practice, deliver new therapeutic targets, and open new directions in molecular investigations of PSU.